A new method for two-dimensional analysis of DNA is under development. The DNA under study is first cleaved with a restriction enzyme; the cleavage products are separated by electrophoresis on specially treated agarose under conditions which permit a second restriction enzyme easy access to the separated fragments. After a suitable incubation, the fragments cleaved by the first enzyme are cleaved again, this time at new sites specified by the second restriction enzyme. The electrophoresis is repeated, but in a direction perpendicular to the first run. The result is that the number of DNA fragments that can be resolved is approximately the square of the number that can be resolved in a one-dimension analysis. The method is expected to be of great utility in locating polymorphic genes and in identifying segments of the gene that may be rearranged during carcinogenesis. The method will also facilitate analysis of heterogeneous nuclear RNA.